Analytical method employing active material contained in a flow-throuh bag

ABSTRACT

A METHOD OF DETERMINING THE PRESENCE OF CERTAIN CONSTITUTENTS IN BIOLOGICAL SAMPLES IN WHICH A BAG, SIMILAR IN CONSTRUCTION TO THE WELL-KNOWN FLOW-THUR TEA BAG, WHICH CONTAINS ONE OR MORE ACTIVE SOLID MATERIALS USED IN ANALYTICAL PROCEDURES,IS USED TOCAPTURE CERTAIN COMPOUNDS PRESENT IN THE BIOLOGICAL PRODUCT, SUCH AS URINE OR BLOOD, SO AS TO CONCENTRATE THE COMPOUND PREFERENTALLY, OR TO BRING ABOUT A PARTICULAR TYPE OF CHEMICAL ACTION SUCH AS HYDROLYSIS, OR FOR CLARIFYING SOLUTIONS.

Patented June 4, 1974 3,814,584 ANALYTICAL METHOD EMPLOYING ACTIVEMATERIAL CONTAINED IN A FLOW-THROUGH BAG Paul M. Tocci, Miami, Fla.,assignor to TLC Corporation, Miami, Fla. N Drawing. Filed Sept. 7, 1972,Ser. No. 286,904 Int. Cl. G01n 33/16 US. Cl. 23-230 B Claims ABSTRACT OFTHE DISCLOSURE A method of determining the presence of certainconstituents in biological samples in which a bag, similar inconstruction to the well-known flow-thru tea bag, which contains one ormore active solid materials used in analytical procedures, is used tocapture certain compounds present in the biological product, such asurine or blood, so as to concentrate the compound preferentially, or tobring about a particular type of chemical act1on such as hydrolysis, orfor clarifying solutions.

This invention relates to a method of determining the presence ofcertain constituents in biological samples. More particularly, theinvention concerns a method for use of a bag, similar in construction tothe well-known flow-thru tea bag, containing one or more active solidmaterials used in analytical procedures. The bags are used, for example,to capture certain molecules or compounds present in biologicalproducts, especially in animal fluids such as urine or blood, so as toconcentrate the compounds preferentially. The method may also be used tobring about a particular type of chemical action such as hydrolysis ofglucuronides and sulfates of hormones and drugs in urine prior toextraction, or for clarifying solutions.

The bag is filled with materials such as adsorbants, absorbants,anionic, cationic, or amphoteric ion exchange resins, non-ionic resins,molecular seives, or water insolubilized enzymes attached topoly(acrylamide) polymers, the material depending on the analyticalprocess to be followed later.

The method has time and cost saving advantages over the methodspresently used, such as the use of columns and slurries. The columnsmust be packed with care, and prepared and washed sequentially. They arealso expensive. The slurry method, or the solution method for enzymes,allows for partial or complete loss of material. It is time-consuming towork with methods requiring filtering.

The process of my invention can be carried out completely in thelaboratory, or started by an unskilled operator at the collection point.Thus, between the collection point and the laboratory, the particularchemical species one wishes to measure is being absorbed, adsorbedorionically or physically trapped for later processing by the technicianin the laboratory. The same is true for enzymatic or catalytichydrolysis.

The method of my invention can also be used in conjunction With thecolumn method by packing the bags into the column for furtherprocessing, following the usual procedures.

The bag is made of porous or latticed material such as paper, non-wovenplastic or woven man-made fibers. A weighed amount of the resin or othermaterial of appropriate particle size is sealed in the bag. Suitably, astring with a label is attached to the bag for identifying the same. Thebag with or without label for identifying the sample may be placed in aseparate envelope or packet for protection or for printed instructions.The bag generally is square in shape, although the bottom may containone or more extra folds so as to expand in use, giving greater volume atthe lower end. Other shapes may be used. The bag is small in size, a 2"approximately square bag usually being appropriate. The size of the bagis related to the amount of resin required for the volume of liquidsample to be used in the analysis. The bag is filled approximately fullunless the resin will swell when placed in the liquid.

The ionic or non-ionic resins are widely varied according to theparticular application. Certain types of synthetic resins haveconsiderable exchange capacity. Both cation and anion exchangers can besynthesized which have relatively high capacities for the exchangeableions. New resins can be tailor-made for specific applications requiringcertain chemical and physical characteristics. The ion-exchange resinsare high molecular weight polymers with ionic groups incorporated in thelattice as integral parts of the polymeric structure. Cation exchangerscontain carboxylic, phenolic phosphoric, or sulfonic groups with anequivalent amount of cations. Anion exchangers contain amine groupsincorporated in the polymer lattice, neutralized by an equivalent amountof anions such as chloride, hydroxyl, phosphate or sulfate ions. Thepolymeric portion of the resin has various amounts of cross-linkingwhich affects the solubility.

The general properties of the resin are that it must be sufiicientlycross-linked to insure negligible solubility. It should be sufiicientlyhydrophilic to permit diffusion of ions through the polymer structure ata measurable and usable rate. The structure should be sufiicientlystable so as not to degrade during use. Its density should be greaterthan that of water. The role of particle size in ion-exchangechromatography has been demonstrated to be extremely important. Definiteranges of size can improve results tremendously over use of wide rangesof particle size. Particle size can be sufiiciently large that theparticles will be contained in the bag without contamination of thesample while testing, and at the same time remain small enough thatrapid action occurs with the chemical to be concentrated. These resinsare sold under various trademarks such as Amberlite, Dowex, Sephadex,and the like. Amberlite resins are synthetic, high capacity, cation andanion exchange resins, sold by Rohm and Haas, Philadelphia. Dowexl-X2-Cl is a synthetic strongly basic anion exchange resin containingquaternary ammonium functional groups which are attached to astyrene-divinylbenzene copolymer.

Another use of the process of my invention is to cause hydrolysis ofcompounds such as glucuronides and sulfates of hormones in urine priorto extraction. The Enzacryls (a trademark registered by Koch-Light, Ltd,of Colnbrook, Bucks, England) are water insoluble enzymes on hydrophiliccarriers based on cross-linked polyacrylamides which contain functionalgroups potentially active in enzyme binding.

Diatomaceous earth in its various commercial forms can also be placed inthe bag for clarifying fluids.

The particle size varies with the material and its use. Amberite resinXAD-2 has a particle size in the range of about 0.05 to 0.5 mm.diameter, or a screen size of about 275 to 500 wet mesh US. Standard.

Two or more bags can beused simultaneously. Thus while just resting in acup or beaker for various lengths of time, hydrolysis and extraction ofselected types of compounds is achieved at the same time, i.e., whilethe contents of one bag is hydrolyzing molecules, the other bag ispicking up the products of the hydrolysis. When the determined time isup the bags are removed and processed further.

Although the bags could be reusable many times after the properwashings, this process is especially useful for a one-time use. Cost isat a minimum because of the 3 small amount of resin required, and thetime of the operator which is saved. For practical purposes, the bag isconsidered disposable.

According to the process of my invention, samples are prepared forfurther analysis by the following steps, which is described specificallyfor a urine sample, However, it is understood that the procedure can beadapted to other biological samples, and body fluids such as bloodserum. Following my procedure, a small sample of urine, or otherbiological fluid which may be diluted if necessary, is placed in abeaker or similar container and the pH adjusted to about -6, usingdilute alkali or acid, as necessary. Suitable alkalies are sodiumhydroxide, sodium carbonate, and sodium bicarbonate; suitable acidsinclude hydrochloric or sulfuric acid.

A small bag made of an inert material such as paper, or woven or unwovenplastic, and containing a resin or adsorbent material is immersed in theliquid. A string attachment having an identifying label may be attachedto the bag. The bag is allowed to remain immersed in the liquid for atime sufficient to cause substantial action to take place. The length oftime can be substantially shortened by adequate shaking. The reactionwith or without shaking can be continued until approximatelyquantitative action has taken place.

The aqueous liquid is decanted from the bag, and the chemical isrecovered from the resin by adding an anhydrous solvent such as acetone,methylene dichloride or ethyl acetate. The solution is dried in anysuitable manner. Conveniently, a small bag containing anhydrous sodiumsulfate is immersed in the solvent. The pH of the anhydrous solution isadjusted as necessary, and the solution is then evaporated to drynessand the resulting solid material reconstituted with a solvent in orderto obtain a solution suitable for analysis, for example, by thin layerchromatography.

EXAMPLE 1 Extraction of drugs from urine using a floW-thru bagcontaining resin (1) Place a 20 ml. sample of urine in a 100 ml.container,and adjust to pH 5-6 with a few drops of dilute hydrogenchloride or sodium hydroxide.

(2) Place a moist bag containing 1.8 g. of Amberlite XAD-2 resin intothe urine sample. If the bag appears dry, it must be moistened. This canbe done by immersing the bag in distilled water and hanging it up toallow the excess water to drip 011.

With the bag in the urine at room temperature, shake the container for7-10 minutes, or allow the bag to remain immersed in the urine for 6-8hours with no shaking. Shaking can be done, for example, with amechanical shaker using 1-2" strokes at the rate of 120-200 strokes perminute.

(3) Decant the urine and discard.

(4) Add 15 ml. of acetone to the container with the bag, and swirl orshake for about 5 minutes. Then remove the resin bag. Another bagcontaining 2.0 g. of anhydrous sodium sulfate is added to the acetonesolution to remove residual water. The solution containing the bag isshaken as in step (2) above.

(5) Decant the acetone into another container and adjust the pH to about2-3 by adding one drop of 1% methanolic hydrogen chloride.

(6) Evaporate the solution to dryness at about 6570 C., using a waterbath in a vented hood. When dry, reconstitute the product with about 50ml. of methanol to obtain a solution suitable for thin layerchromatography for the separation of drugs.

Other resins can be used depending on the drug to be identified andother variables. In place of acetone, methylene dichloride or ethylacetate can be used as the anhydrous solvent.

4 EXAMPLE 2 COMPARISON OF RECOVERIES BY BAG METHOD AND COLUMN METHOD,PERCENT RECOVERY FROM LRINE 1.8g. Amber- 1.8 g. XAD-2 lite XAD-Z resin(dry (dry weight) weight in resin in bag 1 x 5 cm. column Morphine 4055-60 Codeine. 90-92 60-65 Methadone. 85-87 -85 Phenobarbit 80-85 20-50Pentobarbital. 98-100 45-60 Amphetamine 98-100 50-50 Doriden 97-10098-100 Amberlite XAD-2 resin is manufactured by Rohm and Haas Company,Philadelphia, Pennsylvania. It is a synthetic insoluble crosslinkedpolystyrene polymer which has the following specifications:

Hard, spherical, water saturated Appearance opaque beads Solids(percent) 51-55 Porosity (ml. pore/ml./bead) 0. 40-0. 45 Surface area(m. /g.-dry basis 290-330 Efieetive size (mm. 0. 30-0. 45 Harmonic meanparticle size (mm). 0 45-0. 60 Average pore diameter (A.) -95 True wetdensity (g./ml.) 1. 03 Skeletal density (g./ml.) 1. 06 Bulk density:

LbsJitfi--- 40-44 G./cc 0. 64-0. 70

EXAMPLE 3 Centrifuge a 10 ml. sample of blood to obtain 4-5 ml. ofplasma or serum. Immerse into the serum for about 10 to 60 minutes asmall bag cointaining about 1 g. of a resin or diatomaceous earth orcharcoal useful for clarifying the solution and cleaning out thechromophers which interfere with certain analytical procedure due to thepresence of too much color. Continue with the usual analyticalprocedure.

Various modifications and variations of the present invention may bemade without departing from the spirit of the discovery or the scope ofthe appended claims.

What is claimed is:

1. In an analytical procedure for determining the presence of certainconstituents in biological samples, the step of immersing one or moreflow-thru bags, each containing a premeasured amount of a solid reactivematerial into an aqueous biological fluid for a time sufiicient forreaction to take place, and then removing the bag from the liquid toeffect a separation of certain of said constituents.

2. The step of claim 1 wherein said solid reactive material is anon-ionic resin for the absorption of water soluble drugs from urine.

3. The step of claim 1 wherein said solid reactive material is adiatomateous earth material useful for clarifying blood serum prior tofurther analysis.

4. The step of claim 1 wherein said solid reactive material is a waterinsoluble enzyme on a hydrophilic carrier based on cross-linkedpoly(acrylamides) which contain functional groups potentially active inenzyme binding.

5. A method for the analysis of urine which comprises the steps of:

(a) adjusting the pH to about 5-6;

(b) immersing in the sample a fiow-thru bag moistened with watercontaining a non-ionic resin for the adsorption of water soluble organicsubstances, and

5 allowing the bag and sample to stand for 6-10 hours without shaking,or for 7-10 minutes with shaking; (c) decanting the aqueous liquid anddiscarding it; (d) extracting the material to be analyzed from saidresin using an anhydrous solvent, and then dehydrating the solution andadjusting it to a pH of about 23;

(e) evaporating said solution to dryness and reconstituting the dryproduct with a solvent to obtain a solution suitable for chromatographicanalysis.

6. The method of claim 5 wherein the resin is a nonionic resin fordrugs.

7. The method of claim 5 wherein the particle size of the resin is inthe range of about 0.05 to 0.5 mm. in diameter, and the screen size isabout 275 to 500 wet mesh US. Standard.

8. The method of claim 5 wherein the solution in step (d) is dehydratedby use of a bag containing anhydrous sodium sulfate.

9. The method of claim 5 using an ionic exchange resin.

10. The method of claim 5 wherein the dry product in step (e) isreconstituted with a solvent suitable for thin layer chromatographicanalysis to determine the presence 10 3,607,093 9/1971 Stone 23-230BOTHER REFERENCES Chemical Engineering, pp. 44-45, Jan. 12, 1970.

15 RONALD E. SERWIN, Primary Examiner US. Cl. X-R-

